Distribution and characterisation of entomopathogenic fungi from Korean soils

We investigated the occurrence of entomopathogenic fungi in 1080 soil samples representing multiple locations and conditions in Korea. Entomopathogenic fungi were isolated from soils using a selective medium containing dodine and antibiotics. Following an initial identification based on morphology, the fungal isolates were more precisely identified by the sequence of their nuclear ribosomal RNA (rRNA) internal transcribed spacer (ITS) regions. As a result, entomopathogenic fungi were found to occur in 32% (342 isolates) of the soil samples studied. The most abundant species were Beauveria spp. (125 isolates) and Metarhizium spp. (82 isolates). Entomopathogenic fungi were more often recovered from natural mountain and riparian soils than from agricultural habitats. The pathogenicity of isolated fungi was evaluated by using wax moth Galleria mellonella L. (Lepidoptera: Pyralidae) larvae. It was determined that 60% (207 isolates) of the isolates were pathogenic using this model. These entomopathogenic fungi may, therefore, have potential use against a variety of agricultural pests. This is the first study of the isolation and distribution of entomopathogenic fungi in representative sampling locations throughout Korea.     

Distribution,occurrence and characterization of entomopathogenic fungi in agricultural soil in the Palestinian area

The occurrence of entomopathogenic fungi was investigated in irrigated vegetable fields and citrus orchards soils, over a nine-month period (April-December 1999),using the Galleria bait method (GBM). Entomopathogenic fungi were found to occur in 33.6% of the soil samples studied, with positive samples yielding 70 fungal isolates, belonging to 20 species from 13 genera. Conidiobolus coronatus was the most frequent and abundant entomopathogenic species recovered, comprising 31.4% of the total number of isolates. Soil pH, soil moisture content and the geographical location had minor or no effect on the isolation of entomopathogenic fungi in the fields studied. On the other hand, organic matter content of soil, and vegetation type were found to significantly affect the occurrence of entomopathogenic fungi in soil habitats, with orchard fields yielding larger numbers of isolates than vegetable fields. Using Koch's postulates the pathogenicity of fungal isolates to Galleria larvae was found to range from 16–100% (mortality rate). Isolates of C. coronatus proved to be the most virulent isolates recovered. The effect of media and temperature on mycelial growth rate, conidial production and conidial germination of six entomopathogenic fungal species (C. coronatus, Entomophaga grylli, Erynia castrans, Hirsutella jonesii, Paecilomyces farinosus and Sporodiniella umbellata) was also studied. Mycelial growth rate, spore production and spore germination were significantly affected by media, temperature and isolates. In view of the present results, C. coronatus appears to be a good candidate for pest control in agricultural soils, as it has a wide tolerance to agricultural practices, has frequently been isolated from both vegetable and orchard fields, and is characterized by high mycelial growth rate, conidial production and conidial germination.This revised version was published online in October 2005 with corrections to the Cover Date.     

Natural occurrence of soil-borne entomopathogenic fungi in the Moroccan Endemic forest of <Emphasis Type="Italic">Argania spinosa</Emphasis> and their pathogenicity to <Emphasis Type="Italic">Ceratitis capitata</Emphasis>

The occurrence and abundance of entomopathogenic fungi were analysed in 203 soil samples of the Moroccan endemic forests of Argania spinosa, the world main refuge of the Mediterranean fruit fly, Ceratitis capitata. Using the Galleria baiting method and selective media, entomopathogenic fungi were isolated from 186 of the 203 (91.62%) soil samples, with only three species found: Beauveria bassiana (Balsamo) Vuillemin, Metarhizium anisopliae (Metschnikoff) Sorokin and Paecilomyces lilacinus (Thom.) Samson. B. bassiana was the most widespread entomopathogenic fungi (90.64%) in the Argan forest whereas M. anisopliae was less common (15.27%) and P. lilacinus was very rare (1.48%). This is the first report of natural occurrence of M. anisopliae and P. lilacinus in Morocco. Furthermore, 118 Moroccan B. bassiana isolates were studied for their pathogenicity to C. capitata and thermotolerance. Most of these autochtonous B. bassiana isolates were virulent (86.44%) to Medfly pupae and tolerant (55.08%) to temperature stress at 45°C for 2 h. Only 60.17% of Moroccan B. bassiana isolates might be considered as highly entomopathogenic and will serve as a source of potential biological control agents to C. capitata. The percentage of thermotolerant and pathogenic B. bassiana to C. capitata were shown to decrease significantly at winter time characterized by low temperatures and absence of any noticeable medfly in the Argan forest. The occurrence, thermotolerance and virulence of B. bassiana isolates to C. capitata seemed to be related to the sampling periods and location. Our data are discussed with respect to fungal ecology and biocontrol potential of B. bassiana isolates in relation to their habitat.     

Persistence and virulence of promising entomopathogenic fungal isolates for use in citrus orchards in South Africa

Thaumatotibia leucotreta (Meyrick) (1912) (Lepidoptera: Tortricidae) is a key economic pest of citrus in South Africa. Although a variety of control options are available, none currently registered target the soil-dwelling life stages of the pest. Three fungal isolates, two Metarhizium anisopliae sensu lato Metschnikoff (Sorokin) isolates (G 11 3 L6 and FCM Ar 23 B3) and one Beauveria bassiana sensu lato Balsamo (Vuillemin) isolate (G Ar 17 B3) have been identified as showing the greatest potential against T. leucotreta soil-dwelling life stages. Since environmental persistence is an important factor in the success of entomopathogenic fungi against soil pests, the isolates, along with two commercial isolates (B. bassiana Eco-Bb® and M. anisopliae ICIPE 69), were subjected to a six-month field persistence trial. Every month after burial of the fungal isolates, inside net bags, in orchard soil, the number of colony forming units (CFUs) per gram of soil was measured and the viability of the conidia was assessed using bioassays. All isolates investigated were capable of persisting in the soil for the duration of the trial period and in addition, were capable of initiating infection in T. leucotreta late fifth instar larvae and subsequent pupae. However, with the exception of the commercial isolates which showed some correlation, no correlation was found between the number of CFUs recorded and percentage T. leucotreta mycosis. Persistence of entomopathogenic fungi in the field is affected by a series of factors, and although the effect of these factors was not measured, the data have provided preliminary evidence towards the persistence capability of these candidate biopesticides against T. leucotreta.     

Investigation of native isolates of entomopathogenic fungi for the biological control of three citrus pests

The pathogenicity of 15 isolates of Beauveria bassiana (Balsamo) Vuillemin, five isolates of Metarhizium anisopliae (Metschnikoff) Sorokin and one isolate of M. flavoviride (Gams and Rozsypal) were tested under laboratory conditions against the subterranean life stages of the citrus pests, Ceratitis rosa Karsch, C. capitata Wiedemann (Diptera: Tephritidae) and Thaumatotibia leucotreta Meyrick. (Lepidoptera: Tortricidae). When these citrus pests were treated with a concentration of 1×10⁷ conidia mL^?1, fungal isolates had a significantly greater effect on the adults of C. rosa and C. capitata than they did on the puparia of these two fruit fly species. Further, C. rosa and C. capitata did not differ significantly in their response to entomopathogenic fungi when adult and pupal mycosis were considered. Depending on fungal isolate, the percentage of T. leucotreta adults which emerged from fungal treated sand ranged from 5 to 60% and the percentage of pupae with visible signs of mycosis ranged from 21 to 93%. The relative virulence of the four most promising fungal isolates, as well as the commercially available B. bassiana product, BroadBand® (Biological Control Products, South Africa), were compared against one another as log-probit regressions of mortality against T. leucotreta which exhibited a dose-dependent response. The estimated LC₅₀ values of the three most virulent B. bassiana isolates ranged from 6.8×10⁵ to 2.1×10⁶ conidia mL^?1, while those of the least pathogenic ranged from 1.6×10⁷ to 3.7×10⁷ conidia mL^?1.     

Natural occurrence of insect pathogenic fungi and insect parasitic nematodes in <Emphasis Type="Italic">Diabrotica virgifera virgifera</Emphasis> populations

The Western Corn Rootworm D. virgifera virgifera Le Conte (Coleoptera: Chrysomelidae), a serious pest of maize, has been recently introduced into Europe. Several approaches for its control are presently under investigation including microbial agents. In order to get information on the role of naturally occurring pathogens in the regulation of Diabrotica populations, we started an investigation in established populations in Hungary, Romania, Serbia, Austria, and Italy in 2005 and 2006. In infested maize fields in Hungary, plants and their root systems were grubbed out and larvae and pupae were collected. Adult D. v. virgifera were collected in Hungary, Austria, Romania, Serbia and Italy. Additionally, the occurrence of entomopathogenic fungi in soils of maize fields was determined using Galleria mellonella and Tenebrio molitor larvae as bait insects. The density of entomopathogenic fungi was obtained by plating soil suspension on selective medium. Metarhizium anisopliae and Beauveria spp. infections were found in 1.4% of field collected larvae, 0.2% of field collected pupae and 0.05% of field collected adults. Whereas natural infections of D. v. virgifera were rarely found, a high density of insect pathogenic fungi was recorded in Hungarian soils. M. anisopliae could be detected in every maize field either using the “bait method” or a “selective medium” method. This is the first report of a natural occurrence of entomoparasitic nematodes (Heterorhabditis sp., Steinernema sp.) in Diabrotica v. virgifera in Europe.     

Distribution,occurrence and characterization of entomopathogenic fungi in agricultural soil in the Palestinian area

The occurrence of entomopathogenic fungi was investigated in irrigated vegetable fields and citrus orchards soils, over a nine-month period (April-December 1999), using the Galleria bait method (GBM). Entomopathogenic fungi were found to occur in 33.6% of the soil samples studied, with positive samples yielding 70 fungal isolates, belonging to 20 species from 13 genera. Conidiobolus coronatus was the most frequent and abundant entomopathogenic species recovered, comprising 31.4% of the total number of isolates. Soil pH, soil moisture content and the geographical location had minor or no effect on the isolation of entomopathogenic fungi in the fields studied. On the other hand, organic matter content of soil, and vegetation type were found to significantly affect the occurrence of entomopathogenic fungi in soil habitats, with orchard fields yielding larger numbers of isolates than vegetable fields. Using Koch's postulates the pathogenicity of fungal isolates to Galleria larvae was found to range from 16-100% (mortality rate). Isolates of C. coronatus proved to be the most virulent isolates recovered. The effect of media and temperature on mycelial growth rate, conidial production and conidial germination of six entomopathogenic fungal species (C. coronatus, Entomophaga grylli, Erynia castrans, Hirsutella jonesii, Paecilomyces farinosus and Sporodiniella umbellata) was also studied. Mycelial growth rate, spore production and spore germination were significantly affected by media, temperature and isolates. In view of the present results, C. coronatus appears to be a good candidate for pest control in agricultural soils, as it has a wide tolerance to agricultural practices, has frequently been isolated from both vegetable and orchard fields, and is characterized by high mycelial growth rate, conidial production and conidial germination.     

Virulence of entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae against red palm weevil,Rhynchophorus ferrugineus (Olivier)

Nineteen different isolates of the entomopathogenic fungi Beauveria bassiana sensu lato (s.l.) and Metarhizium anisopliae s.l. (Ascomycota: Hypocreales), recovered from different soil samples (field crops, fruit orchards, vegetable fields and forests) and insect cadavers were tested against Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae) at two different spore concentrations (1 × 10⁷ and 1 × 10⁸ conidia mL^?1). Three isolates of B. bassiana and two of M. anisopliae gave >88 % larval and >75 % adult mortality of R. ferrugineus on their highest dose rate respectively. More sporulating cadavers (mycosis) resulted from a high dose rate compared to low dose on both life stages of R. ferrugineus. The current study confirmed the lethal action of B. bassiana and M. anisopliae isolates with mortality levels usually directly proportional to the conidial concentration. This study further confirmed that the isolates recovered from R. ferrugineus dead cadavers gave more mortality compared to the other sources. In the virulence assay two isolates of B. bassiana caused the highest percentage of both larval and adult mortality at all exposure intervals which suggest that they may be the most promising for use in sustainable management programs aimed at microbial control in date palm orchards.     

Isolation and characterization of entomopathogenic fungi from hazelnut-growing region of Turkey

Although Turkey is the first among all hazelnut-producing countries, yield per unit area of this crop is low in comparison to other countries, mainly because many insect species seriously damage hazelnut trees and their fruit. To find effective and safe biocontrol agents, we conducted a survey study to isolate entomopathogenic fungi from the hazelnut-growing region of Turkey and characterized the isolated strains in detail. In addition, we determined the effectiveness of seven selected strains from this region against Melolontha melolontha (Coleoptera: Scarabaeidae) which is one of the most serious pests of hazelnut. In 2006 and 2007, 301 soil samples were collected randomly and analyzed for presence of entomopathogenic fungi using the Galleria bait method. Entomopathogenic fungi were found to occur in 20.59% of the soil samples studied. Based on morphology, ITS sequence and partial sequencing of the 18S (SSU rDNA) and EF1-α genes, the isolates were identified as Metarhizium anisopliae var. anisopliae, Metarhizium sp., Beauveria bassiana, Beauveria cf. bassiana, Isaria fumosorosea and Evlachovaea sp. Metarhizium anisopliae var. anisopliae was isolated from 34 sites and was the most frequent and abundant entomopathogenic species recovered. All the isolates tested were pathogenic to M. melolontha. M. anisopliae var. anisopliae KTU-27 and Evlachovaea sp. KTU-36 produced the highest insecticidal activity (86.6%) within 15 days after inoculation. Our results suggest that entomopathogenic fungi could be good biocontrol agents against M. melolontha, and are discussed with respect to ecology of fungi in relation to habitat in order to evaluate biocontrol potential of these isolates. This is the first study of the distribution of entomopathogenic fungi in the hazelnut-growing region of Turkey and of their pathogenicities against M. melolontha.     

Occurrence and diversity of entomopathogenic fungi in cultivated and uncultivated soils in Pakistan

The distribution of entomopathogenic fungi in various geographical areas of Punjab, Pakistan, is poorly understood. The present study was planned to explore the occurrence and diversity of entomopathogenic fungi from soils collected from cultivated and non‐cultivated habitats. The detailed survey of different habitats (crop fields, fruits, vegetables, forests) was conducted to collect soil samples and the associated fungi were isolated using Galleria bait method. Among 210 soil samples, 168 fungal isolates were recovered and identified, with 98 from forests, 32 from vegetables, 30 from field crops and 8 from fruits. The major entomopathogenic fungi recovered from these samples were Beauveria bassiana, Metarhizium anisopliae, Paecilomyces lilacinus, B. brongniartii, P. chlamydosporia and Lecanicillium attenuatum. The diversity of entomopathogenic fungi was greater in soil samples from forests compared to crop fields, vegetables and fruits, respectively. The geographical attributes (altitude, longitude, latitude) greatly influenced the occurrence of entomopathogenic fungi with the highest number of isolates found from >600 m altitude, 33°–34′N latitude, and 73°74′E longitude from soil samples. The results of the surveys clearly indicated that the entomopathogenic fungi are distributed in the soils which may be used in successful IPM programs in future.     

Niche separation of species of entomopathogenic fungi within the genera Metarhizium and Beauveria in different cropping systems in Mexico

Entomopathogenic fungi from the genera Beauveria and Metarhizium, were isolated from soil using the Galleria mellonella baiting method, and from infected white grub larvae from a diversity of cropping systems in Puebla and Guanajuato, Mexico. Isolates were identified to species level using Bloc and Elongation Factor 1-α sequence information. Although widespread, Beauveria bassiana (41 isolates) was only isolated from soil and not from infected white grubs. In contrast, Beauveria pseudobassiana (six isolates) was predominantly isolated from white grub larvae (only one isolate from soil). Haplotype analysis of B. bassiana Bloc sequences identified 25 haplotypes indicating substantial genetic diversity; neither geographical origin nor crop type explained this genetic variation. Metarhizium brunneum (three isolates) and Metarhizium robertsii (17 isolates) were also only isolated from soil, while Metarhizium anisopliae (six isolates) and Metarhizium pingshaense (four isolates) were only isolated from white grub larvae. M. anisopliae was only found infecting Paranomala species while M. pingshaense was only found infecting Phyllophaga species. Species diversity in Metarhizium was influenced by crop type. Our results showed that entomopathogenic fungi species could co-exist in the same soil ecosystem but in separate niches. The potential ecological roles of these species are discussed.     

Comparative study of entomopathogenic nematode isolation using Galleria mellonella (Pyralidae) and Spodoptera littoralis (Noctuidae) as baits

Entomopathogenic nematode (EPN) occurrence in soil from natural areas and crop field edges from La Rioja (northern Spain) was compared using two insects as baits: Galleria mellonella (Lepidoptera: Pyralidae) and Spodoptera littoralis (Lepidoptera: Noctuidae). Both insects trapped Steinernema feltiae, S. kraussei and S. carpocapsae, with G. mellonella being more efficient than S. littoralis recording 5.4 and 2.6% of positive soil samples, respectively. EPN recovery frequency and abundance obtained with G. mellonella were not statistically different between natural and crop field edges values; however, S. littoralis was more successful trapping EPNs from crop field edges. Statistical differences were observed for recovery frequency recorded by both hosts in natural areas. Significant differences in larval mortality between both insects were not observed. The use of S. littoralis in entomopathogenic nematode surveys is discussed.     

Efficacy of soil application of Metarhizium anisopliae and the use of GF-120 spinosad bait spray for suppression of Bactrocera invadens (Diptera: Tephritidae) in mango orchards

Field trials were conducted for two seasons in mango orchards at Nthagaiya, Kenya, to evaluate the efficacy of soil inoculation with Metarhizium anisopliae (Hypocreales: Clavicipitaceae) and GF-120 spinosad bait sprays, applied either alone or in combination, for suppression of the invasive fruit fly, Bactrocera invadens (Diptera: Tephritidae). During the 2006/2007 season, average post-treatment samples showed that B. invadens catches from the control orchards were four times higher than the number of flies captured in the plots receiving M. anisopliae+GF-120. Fruit infestation was 16, 45, 30 and 60% in the M. anisopliae+GF-120, M. anisopliae alone, GF-120 alone and control orchards, respectively. In the 2007/2008 season, average B. invadens post-treatment samples in the control orchards were seven times higher than the treatment with M. anisopliae+GF-120; and fruit infestation was 11, 38, 28, and 52% in the orchards assigned to M. anisopliae+GF-120, M. anisopliae alone, GF-120 alone and control treatments, respectively. Enumeration of conidial densities from soil samples on agar plates showed initial densities of 1.1–2.1×10⁵ colony forming units (cfu)/g of dry weight of soil but decreased to 1.0–1.4×10³ cfu/g at the end of the experimental period. Exposure of laboratory-reared pupariating larvae to soil samples taken from fungus-treated fields showed significant reduction in B. invadens adult emergence (25–36%) compared with the control (80–82%). Our results suggest that the combined use of soil application of M. anisopliae and GF-120 spinosad bait spray is an effective IPM strategy for field suppression of B. invadens on mango.     

Change in the temperature preferences of <Emphasis Type="Italic">Beauveria bassiana sensu lato</Emphasis> isolates in the latitude gradient of Siberia and Kazakhstan

The radial growth of twenty isolates of the entomopathogenic fungus Beauveria bassiana sensu lato from different natural zones of Western Siberia and Kazakhstan (from 65 to 43°N) was tested under different temperatures (5–35°C). It was shown that the thermotolerance of the fungal isolates increased significantly from the north to south. The cold activity of the cultures did not significantly correlate with the latitude of origin and the sum positive temperatures of the regions. A distinct group of the steppe thermotolerance isolates was shown by the analysis of genomic polymorphism using seven intermicrosatellite DNA markers (ISSR). The steppe isolates had high levels of virulence to the wax moth Galleria mellonella and the Colorado potato beetle Leptinotarsa decemlineata at high temperatures (>30°C) compared to that of the forest-steppe isolates. The obtained data indicate that the use of isolates from the steppe zone will be most promising for the insect pest control under the conditions of continental and arid climate.     

Variability in virulence of Beauveria spp. soil isolates against Ostrinia nubilalis

European corn borer (Ostrinia nubilalis) is an important maize pest which is treated mainly by synthetic pesticides with many negative effects for ecosystem and human health. Soil borne entomopathogenic fungi of species belonging to Beauveria genus may provide an alternative strategy for biological control of this pest. In this article, we report results of virulence testing of 46 soil isolates of Beauveria spp. against O. nubilalis in relation to isolates origin and haplotype. All strains were collected from soil using Galleria mellonella bait method. Conidia suspension with concentration 10⁷ conidia in ml was used for virulence test against 4th instar larvae of O. nubilalis at temperature 25°C. After 14 days, mortality of larvae was in the range 34%–96%. Median lethal time LT50 was estimated to 5.5–21.3 days. Mortality was not in relation to habitat or any other environmental parameter of sites where isolates came from. Differences among species were insignificant. Isolates were divided into 8 genetic lineages and 14 haplotypes using sequencing of ITS and Bloc nuclear regions. None of the lineages showed higher mortality than others. Despite more or less virulent isolates being found in each lineage, the three most virulent isolates belonged to the same lineage Beauveria bassiana A3. The four most virulent isolates were compared to commercial isolate GHA to assess medium lethal concentration. LC50 were estimated in range 7.5 × 10⁴ to 9.5 × 10⁵. Three of the four isolates showed equal or greater efficiency than strain GHA.     

Attract‐and‐kill systems efficiency against Ceratitis capitata (Diptera: Tephritidae) and effects on non‐target insects in peach orchards

For control of the Mediterranean fruit fly Ceratitis capitata (Diptera: Tephritidae), the attract‐and‐kill or attracticide technique is an alternative to the spraying of traditional organophosphate pesticides. In this study, the effectiveness of Ceranock and AAL&K attract‐and‐kill bait stations was assessed for control of C. capitata in Tunisian peach (Prunus persica) orchards. Our results showed that, in orchards with early‐ripening varieties, the numbers of C. capitata males and fruit damage were significantly lower in plots treated with Ceranock and AAL&K bait stations than in plots treated with conventional organophosphate and pyrethroid insecticides. In addition, the abundances of non‐target insects in the Chrysopidae, Coccinellidae and Miridae were significantly greater in plots treated with the bait stations than in plots treated with the conventional pesticides; that is, the use of attract‐and‐kill bait stations had fewer negative effects than the application of conventional pesticides on the biological diversity in Tunisian peach orchards. Overall, the results indicate that Ceranock and AAL&K attract‐and‐kill bait stations are useful alternatives for the control of C. capitata in Tunisian peach orchards planted with early‐ripening varieties.     

Factors affecting the occurrence and distribution of entomopathogenic fungi in natural and cultivated soils

Factors affecting the occurrence and distribution of entomopathogenic fungi in 244 soil samples collected from natural and cultivated areas in Spain were studied using an integrated approach based on univariate and multivariate analyses. Entomopathogenic fungi were isolated from 175 of the 244 (71.7 %) soil samples, with only two species found, Beauveria bassiana and Metarhizium anisopliae. Of the 244 soil samples, 104 yielded B. bassiana (42.6 %), 18 yielded M. anisopliae (7.3 %), and 53 soil samples (21.7 %) harboured both fungi. Log-linear models indicated no significant effect of habitat on the occurrence of B. bassiana, but a strong association between M. anisopliae and soils from cultivated habitats, particularly field crops. Also, irrespective of habitat type, B. bassiana predominated over M. anisopliae in soils with a higher clay content, higher pH, and lower organic matter content. Logistic regression analyses showed that pH and clay content were predictive variables for the occurrence of B. bassiana, whereas organic matter content was the predictive variable for M. anisopliae. Also, latitude and longitude predicted the occurrence of these same species, but in opposite directions. Altitude was found to be predictive for the occurrence of B. bassiana. Using principal component analysis, four factors (1 to 4) accounted for 86 % of the total variance; 32.8, 22.9, 19.6 and 10.4 % of the cumulative variance explained, respectively. Factor 1 was associated with high positive weights for soil clay and silt content and high negative weights for soil sand content. Factor 2 was associated with high positive weights for soil organic matter content and high negative weights for soil pH. Factor 3 was associated with high positive weights for latitude and longitude of the sampled localities and factor 4, had high positive weights only for the altitude. Bi-plot displays representing soil samples were developed for different factor combinations and indicated that, irrespective of geographical location, absence of both fungal species was determined by alkaline sandy soils with low organic matter content, whereas heaviness of soil texture, acidity and increasing organic matter content led to progressively higher percentages of samples harbouring entomopathogenic fungi. These results could aid decision-making as to whether or not a particular cultivated or natural soil is suitable for using entomopathogenic fungi as a pest control measure and for selecting the fungal species best suited to a particular soil.     

Comparison of the relative efficacy of an insect baiting method and selective media for diversity studies of Metarhizium species in the soil

Metarhizium are a commonly occurring group of entomopathogenic fungi normally found in soil. The most common methods to assess the diversity of Metarhizium species in soil are (i) the use of selective media and (ii) insect baiting using Galleria mellonella larvae. We compared the recovery efficiency from soil of four common species of Metarhizium (Metarhizium anisopliae, Metarhizium pingshaense, Metarhizium brunneum and Metarhizium robertsii) using these two methods. Firstly, we compared the number of colony forming units (CFU) produced in vitro when grown on two selective media, one containing chloramphenicol, thiabendazole and cycloheximidethe (CTC) and one based on the fungicide dodine (n-dodecylguanidine acetate) (DOD). Secondly, we artificially inoculated natural/non-sterile soil with the four fungal species at a rate of 2×10² and 2×10³ conidia g^?1of soil, baited with G. mellonella, and processed for evaluation using the selective media. The in vitro results showed that the greatest number of CFUs were recorded for M. brunneum. In contrast, when inoculated into soil, more G. mellonella larvae became infected by M. anisopliae. Finally, when using selective media, most CFUs recovered were for M. robertsii. The importance of our results in selecting a method to study the natural occurrence of Metarhizium in soil are discussed.     

Relative susceptibility of <Emphasis Type="Italic">Spodoptera litura</Emphasis> pupae to selected entomopathogenic fungi

The pupae of Spodoptera litura (Fab.), (Lepidoptera: Noctuidae), a polyphagous pest affecting common crops in Indian subcontinent, were treated with different concentrations of conidia of four isolates of entomopathogenic fungi belonging to three species, Metarhizium anisopliae var. anisopliae (Metschnikov) Sorokin (ARSEF 7487), Lecanicillium muscarium (Petch) Zare & W Gams (two isolates ARSEF 7037 and ARSEF 6118) and Cordyceps cardinalis Sung & Spatafora (ARSEF 7193) under laboratory conditions. Suspensions (10⁸/ml) of conidia harvested from Sabouraud dextrose agar yeast extract (SDAY) plates resulted in the highest mortality (85.8%) with M. anisopliae and the lowest mortality (57.3%) with C. cardinalis. The values of LC₅₀ and LC₉₀ suggested that M. anisopliae was the most virulent fungal strain followed by L. muscarium (ARSEF 7037). However, C. cardinalis was the least virulent species among the fungi used in the bioassay. In soil bioassays, drenching the soil with conidial suspensions of ARSEF 7487 and ARSEF 7037 (10⁸ conidia/g of soil) reduced the adult emergence from pupa by 81.3% and 72.5%, respectively, while premixing the sterile soil with conidia killed lesser number of pupae (62.9% by ARSEF 7487 and 54.6% by ARSEF 7037). Our findings suggest that M. anisopliae (ARSEF 7487) and L. muscarium (ARSEF 7037) are potent entomopathogens and could be developed into biocontrol agents against rice cutworm in IPM programs. Handling editor: Helen Roy     

Culture conditions affect virulence and production of insect toxic proteins in the entomopathogenic fungus Metarhizium anisopliae

Conidial spores are often used as the infectious agent during insect biocontrol applications of entomopathogenic fungi. Here we show differential virulence of conidia derived from Metarhizium anisopliae strain EAMa 01/58-Su depending upon the solid substrata used for cultivation, where LC₅₀ values differed by up to ~10-fold (5.3×10⁶?4.5×10⁵ conidia/ml) and LT₅₀ values by ~40% (9.8?7.1 d). This fungal strain is also known to secrete proteins that are toxic towards adult Mediterranean fruit flies, Ceratitis capitata, and the Greater wax moth, Galleria mellonella, larvae. In vitro production and intrahemoceol injection using G. mellonella as the host was used to test fractions during purification of the protein toxins, demonstrating that they elicited defence-related responses including melanisation and tissue necrosis. Production of these proteins/peptides along with a number of potential cuticle degrading enzymes was confirmed both in vitro and during the infection process (in vivo). Two-dimensional gel electrophoresis, followed by gel elution and bioassay, was used to identify at least three proteins or peptides (molecular mass=11, 15 and 15 kDa) as mediating the observed insect toxicity. These data demonstrate that in vitro screening for insect toxins can mimic in vivo (i.e. during the infection process) secretion and applies the use of proteomics to invertebrate pathology.